PROJECT SUMMARY Botulinum neurotoxins (BoNTs) are among the most toxic agents known to humans and cause the life threatening, neuroparalytic disorder botulism. The potential for major public health impact resulting for an intentional release, combined with the paucity of approved vaccines or therapies has led to the classification of BoNTs as Tier 1, Category A Select Agents. Paradoxically, the highly specific action of BoNTs make them excellent therapeutics for a growing and heterogeneous number of human diseases that are characterized by a hyperactivity of peripheral nerve terminals. Despite many recent advances in understanding the structure- function relationship of BoNTs, the molecular events by which the neurotoxin heavy chain (HC) is able to translocate the light chain (LC) across the membrane of endocytic vesicles remains poorly defined. Understanding the mechanism of pH-driven neurotoxin unfolding and translocation is not only of intrinsic value, but also addresses general biophysical questions underlying membrane protein assembly and stability. Site- selective fluorescence labeling of neurotoxins in conjunction with an array of biochemical, spectroscopic and molecular approaches will be employed to test the central hypothesis stating that membrane insertion of BoNT is a regulated process containing key intermediate states which precede formation of the protein translocating channel. Aim 1 will determine the contribution of the receptor binding (HCR) domain in formation of the membrane inserted channel. Specifically, aim 1 will test the hypothesis that the HCR domain functions as a sensor of environmental pH and membrane composition which ensures channel formation occurs at the correct site and time. Aim 2 will address the hypothesis that formation of the BoNT/A channel occurs through a series of interfacial intermediate states. Completion of the proposed studies will provide opportunities for the development of post-exposure therapeutics and improved pharmacologic agents for the treatment of neuronal disorders.